Highly specialized and partially redundant mechanisms have evolved to maintain genomic stability. New evidence suggests that one of these special caretaker mechanisms is defective in Fanconi anemia, a genetic model for human host susceptibility to cancer. FA is a rare but devastating multi-gene disease thought to have an underlying defect in DNA interstrand crosslink repair. Current models suggest that a trigger point for the FA pathway is the activation of a downstream protein, FANCD2. A set of at least eight functionally-interdependent FA core complex proteins is indispensible for the function of FANCD2. The identity and timing of modification events, and how the core complex mediates the enigmatic downstream function of FANCD2 is unclear. Emerging evidence links FANCD2 to DNA repair proteins BRCA1 and BRCA2 (also known as FA protein FANCD1), and a role in the DNA damage response during S-phase. To dissect the key events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. These experimentally tractable extracts include abundant nuclear proteins, abundant replicating chromatin, precise cell cycle synchronization, and naturally-regulated cell cycle control. Coupled with the ability to immunodeplete target proteins (even those that are essential) followed by adding back mutant proteins, these extracts are unique and powerful tools for elucidating the mechanistic role of FA pathway proteins. Our preliminary results demonstrate highly-regulated association of xFANCD2 and core complex proteins with chromatin in S-phase and provide a platform for a sequential model for FANCD2 and core complex protein function. Our central hypothesis is that activated FANCD2 functions within the context of DNA replication to resolve damage. Our aims are: (1) Examine the connection between DNA-damage induced modification(s) and chromatin binding of xFANCD2 by identifying the modifications present on chromatin-bound FANCD2 by biochemical methods and mass spectrometry. FANCD2 mutant proteins will be used to determine which modifications are required for FANCD2 chromatin binding. (2) Characterize chromatin binding of the FA core complex protein xFANCA in the DNA damage response. We will determine if chromatin binding of xFANCA binds to chromatin before or after xFANCD2 in normal and DNA-damaged extracts and determine if chromatin binding of xFANCA and xFANCD2 are interdependent. (3) Examine the function of FANCD2 during replication. We will examine replication in the presence and absence of XFANCD2 to determine if it is required for normal progress of DNA replication, or removal of DNA double strand breaks that arise naturally during replication.